l alpha, 25-Dihydroxyvitamin D3 (l alpha, 25-(OH)2-D3) is a sterol formed in the kidney which is the hormonal form of vitamin D. Studies will be directed toward elucidating the molecular mode of action of l alpha, 25-(OH)2-D3) in stimulating calcium transport across the intestine. Cytoplasmic and nuclear receptors for the hormone will be isolated and characterized in terms of their interrelationship, and the effects of the chromatin-bound nuclear form on template efficiency and RNA polymerase activity will be examined to delineate how the hormone may be altering DNA transcription. A second objective will be the clarification of the endocrine loop which regulates the secretion of l alpha, 25-(OH)2-D3 from the kidney. The renal enzyme catalyzing hormone synthesis, namely 25-hydroxyvitamin D3-l alpha-hydroxylase, will be investigated via four strategies: 1) isolation of the enzyme from chick, 2) assay of the enzyme (in vitro) after various physiologic challenges to the chick, 3) competitive protein binding assay of circulating levels of hormone in rats and pigs manipulated with diets and by surgical removal of glands eliciting endocrine principles which may regulate l alpha, 25-(OH)2-D3 formation, and 4) growth of renal cells in tissue culture and assay of the enzyme in the presence and absence of suspected controlling factors. Finally, we plan to use a newly developed radioreceptor assay for l alpha, 25-(OH)2-D3 in humans to study the possible involvement of this hormone in the etiology of metabolic bone diseases such as renal osteodystrophy, hypoparathyroidism, hyperparathyroidism, sarcoidosis, idiopathic hypercalcemia, vitamin D-resistant rickets, osteoporosis, neonatal hypocalcemia, etc. Patients found to have abnormally low circulating hormone will be considered candidates for treatment with l alpha, 25-(OH)2-D3. Correlation of improved calcium homeostasis and bone integrity with increases in plasma l alpha, 25-(OH)2-D3 concentration will be made.